Statistical optimization of P(3HB-co-3HHx) copolymers production by Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp and its properties characterization

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] is a bacterial copolymer in the polyhydroxyalkanoates (PHAs) family, a next-generation bioplastic. Our research team recently engineered a newly P(3HB-co-3HHx)-producing bacterial strain, Cupriavidus necator PHB−4/pBBR_CnPro-phaCRp. This strain can produce P(3HB-co-2 mol% 3HHx) using crude palm kernel oil (CPKO) as a sole carbon substrate. However, the improvement of P(3HB-co-3HHx) copolymer production by this strain has not been studied so far. Thus, this study aims to enhance the production of P(3HB-co-3HHx) copolymers containing higher 3HHx monomer compositions using response surface methodology (RSM). Three significant factors for P(3HB-co-3HHx) copolymers production, i.e., CPKO concentration, sodium hexanoate concentration, and cultivation time, were studied in the flask scale. As a result, a maximum of 3.6 ± 0.4 g/L of P(3HB-co-3HHx) with 4 mol% 3HHx compositions was obtained using the RSM optimized condition. Likewise, the higher 3HHx monomer composition (5 mol%) was obtained when scaling up the fermentation in a 10L-stirrer bioreactor. Furthermore, the produced polymer's properties were similar to marketable P(3HB-co-3HHx), making this polymer suitable for a wide range of applications.


Results
Optimization of P(3HB-co-3HHx) production by RSM. The optimal medium composition and cultivation condition for P(3HB-co-3HHx) production and the interaction effects of each parameter were determined using a three-variable-five-level CCD design. The CCD variables included CPKO concentration, g/L(X1), sodium hexanoate concentration, g/L(X2) and culture time, h(X3). The experimental results of P(3HB-co-3HHx) production and predicted responses are demonstrated in Table 1. The results revealed that the highest P(3HBco-3HHx) production (Run 6), 3.54 g/L, was achieved when the concentration of CPKO, sodium hexanoate, and cultivation time were 15 g/L, 1.0 g/L, and 54 h, respectively. While the lowest P(3HB-co-3HHx) production (Run 3) was 0.53 g/L when the concentrations of CPKO, sodium hexanoate, and cultivation time were 5 g/L, 3.0 g/L, and 42 h, respectively. The CCD experiment outputs from multiple regression analyses were fitted to a second- www.nature.com/scientificreports/ order polynomial model. The following model was employed to fit P(3HB-co-3HHx) production in terms of coded variables.
where Y is the P(3HB-co-3HHx) production and X1, X2, and X3 are coded values of CPKO, sodium hexanoate, and cultivation time, respectively. The F test and ANOVA for the response surface quadratic model confirmed the equation's statistical significance. R 2 = 0.9885 was the determination coefficient in this study's regression equation ( Table 2). As a result, this model can account for approximately 98.85% of the variability in the dependent variable; the remaining 1.15% was influenced by other factors. While the modified R 2 , which considers the sample size and number of terms 25 , was 0.9782. R 2 values are constantly between 0 and 1. The higher the R 2 , the more influential the model and the better it predicts the response 21 . P-values are used to assess the significance of each coefficient, which contributes to understanding the pattern of mutual interactions among the variables 26 . The stronger the significance of the corresponding coefficient 27 , the smaller the P value. The F test and the corresponding P-values were estimated, as shown in Table 2. The model indicates that the constant linear (X1, X2, X3), quadratic (X1 2 , X2 2 , X3 2 ), and interaction terms (X1X3 and X2X3) are significant (P < 0.05) ( Table 2). However, due to the P-value for all variables (X1, X2 and X3) was smaller than 0.0001, it barely indicates which variables are the most significant for P(3HB-co-3HHx) production.
The negative polynomial coefficient in interaction terms in this model suggests that the interaction is oppositional. The lack of fit F-value of 1.26 (Table 2) indicates that the lack of fit is not statistically significant compared to the standard error. This high lack of fit F-value has a 40.36 percent probability of occurring due to noise.
According to the RSM 3D graphs and 2D contour plots of CPKO(X1) and cultivation time(X2) on P(3HBco-3HHx) production (Fig. 1B), it was shown that the P(3HB-co-3HHx) production significantly improved when CPKO was increased from 5.0 to 15.0 g/L. At the same time, it was reduced when the CPKO concentration declined to below 5.0 g/L. Besides, the production of P(3HB-co-3HHx) rose when the cultivation time was down from 54 to 42 h. Nevertheless, as cultivation time increased (> 54 h), P(3HB-co-3HHx) production dramatically decreased.
The effect of sodium hexanoate and cultivation time are shown in Fig. 1C. P(3HB-co-3HHx) production increased with decreased sodium hexanoate, from 3.0 to 1.0 g/L. While P(3HB-co-3HHx) production dramatically decreased at a higher concentration of sodium hexanoate (> 3.0 g/L) and increased with extended time, from 42 to 54 h. In addition, P(3HB-co-3HHx) production declined when cultivation time was lesser than 42 h.
The model was verified for the three factors in the design space to validate the optimization predictions. RSM-optimized medium composition and conditions were tested in triplicate on a 250-flask scale. The results demonstrate that under the following conditions: CPKO, 14.4 g/L, sodium hexanoate, 1.7 g/L, and 43 h of cultivation time, the maximum P(3HB-co-3HHx) production of 3.63 ± 0.4, with 5.54 ± 0.8 g/L of DCW was obtained, www.nature.com/scientificreports/ nearing the predicted P(3HB-co-3HHx) production of 3.55 g/L. The predicted and experimental values were compared, and the residual was calculated. The relative difference between the actual and predicted P(3HB-co-3HHx) production levels was 0.3%. As a result, the observed models are very accurate, and RSM analysis is an appropriate approach for predicting and improving fermentation medium and conditions.  www.nature.com/scientificreports/ Scaling up of P(3HB-co-3HHx) production in a 10L bioreactor. Batch cultivation was carried out in a 10L stirred-tank bioreactor to enhance the cell biomass and P(3HB-co-3HHx) production of C. necator PHB − 4/pBBR_CnPro-phaC Rp . The fermentation was carried out in a bioreactor containing 6 L of RSM-optimized media (CPKO, 14.4 g/L, sodium hexanoate, 1.7 g/L). The temperature, pH, aeration rate, and agitation speed were fixed at 30 °C, 6.8, 0.25 vvm, and 200 rpm, respectively. The growth and P(3HB-co-3HHx) production of C. necator PHB − 4/pBBR_CnPro-phaC Rp slowly increased during 48 h fermentation. As seen in Fig. 2, biomass increased gradually over fermentation. However, when the fermentation period was extended above the optimum (42 h), P(3HB-co-3HHx) production and cell growth were interrupted and the degradation of P(3HBco-3HHx) began 28 . The highest production of P(3HB-co-3HHx) was at 42 h when the DCW was 6.2 ± 0.3 g/L; P(3HB-co-3HHx) production was 3.9 ± 0.3 g/L (Fig. 2). Besides, it should be noted that the higher 3HHx monomers fraction (5 mol%) was obtained when culturing C. necator PHB − 4/pBBR_CnPro-phaC Rp in a 10L stirredtank bioreactor.
P(3HB-co-3HHx) copolymer characterization. The extracted P(3HB-co-5 mol% 3HHx) copolymer produced from C. necator PHB − 4/pBBR_CnPro-phaC Rp was characterized by 1 H NMR, FTIR, DSC and TGA to understand the copolymer's structural and thermal characteristics for further applications. The 1 H NMR was performed to verify the presence of 3HHx monomer in the copolymer synthesized by the strain C. necator PHB − 4/pBBR_CnPro-phaC Rp . Figure 3 illustrates the 1 H NMR band of H4, corresponding to the C4 methylene groups, and the 1 H NMR band of H6, corresponding to the C6 methyl group, indicating the formation of the P(3HB-co-3HHx) copolymer 15,24,29 . The monomer fractions of the copolymer were calculated according to the 1 H spectrum intensity ratio of the methyl components 30 . The values of the 3HHx monomer fractions produced were slightly higher than those observed by gas GC analysis, with a variation of 1 mol%. FTIR absorption spectra were scanned in the range of 4000-400 cm −1 . The FTIR spectra of copolymer synthesized by C. necator PHB − 4/pBBR_CnPro-phaC Rp are depicted in Fig. 4. The main absorption peak of P(3HBco-3HHx) was observed in the spectrum at 1720.98 cm −1 , which corresponds to the stretching vibration of the carbonyl (C=O) ester bond 31,32 . While the asymmetric C-O-C stretching vibration causes the absorption peak at 1269.35 cm − 1 33 . The C-H stretching and the -CH group were represented by the other distinctive bands located at 2976.37 cm −1 and 1221.72-1375.09 cm −1 , respectively 34,35 . For the amorphous phase, the C-O and C-C stretching vibrations were attributed to a series of absorption bands ranging from 1179.79 to 606.08 cm −133 .
Thermal properties of the P(3HB-co-5 mol% 3HHx) copolymer produced by C. necator PHB − 4/pBBR_CnPro-phaC Rp were analyzed using DSC and TGA. Figure 5 demonstrates the thermogram of melting temperature (T m ), glass transition temperature (T g ), while, and Fig. 6 shows the degradation temperatures (T d ) of the copolymer. The values were recorded from the second heating to eliminate the thermal history of the previous samples. The thermogram of extracted copolymer revealed two melting temperatures (T m1 and. T m2 ) at approximately 129 and 144 °C (Fig. 5). The T c , T g and T d of the copolymer were around 89, 1.6 ( Fig. 5) and 260.6 °C (Fig. 6), respectively.

Discussion
PHAs are biodegradable polymeric materials produced from bacteria and archaea under stress conditions such as nutrient-limiting concentrations of nitrogen, phosphorus, sulfur, or oxygen and excess carbon sources 7,8,36 . Apparently, PHAs are becoming an increasingly significant issue in the scientific community as well as the business sector. This makes it possible for synthetic polymers to be replenished, ultimately establishing the intended circular economy. P(3HB-co-3HHx) is a practical type of PHA copolymer. It has a lower melting temperature and crystallinity than P(3HB) homopolymer, which can be attributed to the 3-hydroxyhexanate (3HHx) unit's long side chain 37 . The copolymers are composed of 5-15 mol% 3HHx and have elastic properties that make them suitable for various applications 38 .
Previously, Han et al. reported that the wild-type strains, Aeromonas spp., can produce P(3HB-co-3HHx) from vegetable oils and fatty acids via PHA synthase, which has a uniquely broad substrate specificity to (R)-3-hydroxyacyl-CoAs of C4-C6 39 . In this study, the P(3HB-co-3HHx) copolymer was synthesized by C. necator   www.nature.com/scientificreports/ PHB − 4/pBBR_CnPro-phaC Rp 23,24 , a newly engineered bacterial strain, and the P(3HB-co-3HHx) production was improved using RSM. The results showed that the highest 3.6 ± 0.4 g/L of P(3HB-co-4 mol% 3HHx) was obtained using the RSM-optimized conditions in the flask scale. Besides, the 3HHx monomer composition was increased to 5 mol% when cultivation C. necator PHB − 4/pBBR_CnPro-phaC Rp in the 10L bioreactor during 48 h of fermentation. This result was similar to the result achieved by Ouyang et al. 40 . They carried out the shake-flask experiments to produce P(3HB-co-3HHx) from Aeromonas hydrophila, which controllable monomer composition from 15% in the wild type to 3-12% in the recombinant by basically changing the ratio of gluconate to lauric acid in the culture media in 48 h of fermentation. The P(3HB-co-3HHx) production studied in Cupriavidus sp. has been reported by Volova et al. 22 , C. eutrophus B10646 could produce significant biomass yields (5.6 g L −1 ), and a high content of the polymer (60-75%), including a high 3HHx molar fraction, under the proper growth conditions. However, the physicochemical and mechanical characteristics of P(3HB-co-3HHx) copolymers can  www.nature.com/scientificreports/ be changed by adjusting the 3HB/3HHx ratio. Besides, Kawashima et al. 41 found that the region downstream of phaP1Re was an advantageous site for integrating genes that are overexpressed during PHA accumulation in R. eutropha. The findings also demonstrated that the polymerization characteristics of PHA synthase were influenced by the kind of phasin that coexisted on the surface of PHA granules, altering the resultant PHA polymer (3HB-co-3HHx). The replacement of phasin is an innovative technical method for controlling the composition of PHA copolyesters. Furthermore, Murugan et al. 42 investigated palm olein (PO) and fructose as carbon sources for the biosynthesis of P(3HB-co-3HHx) by recombinant C. necator Re2058/pCB113. Shake flask cultures utilizing 5 g/L PO as the only carbon source yielded a cell dry weight (CDW) of 5.13 g/L, 67% PHA/CDW, and a copolymer comprising 27 mol% 3HHx. P(3HB-co-3HHx) with 4-15 mol% 3HHx monomer had molecular weights in the range of 5.47-6.85 × 10 5 Da, which was at least two-fold more than previously reported values.
In this study, RSM employing CCD was applied to improve the production of P(3HB-co-3HHx) in a flask scale. The result shows that under the following conditions: CPKO, 14.4 g/L, sodium hexanoate, 1.7 g/L, and 43 h of cultivation time, the maximum P(3HB-co-3HHx) production of 3.63 ± 0.4 was obtained, nearing the RSM predicted P(3HB-co-3HHx) production of 3.55 g/L. These results proved the accuracy of the RSM model for P(3HB-co-3HHx) production. Furthermore, compared to the unoptimized condition, the optimized medium can improve the production of P(3HB-co-3HHx) and 3HHx monomer composition by 1.2 and twofold, respectively. Based on this, the improvement of P(3HB-co-3HHx) production was slightly present, however, the 3HHx monomer composition was successfully enhanced by RSM. Previously, RSM was reported to be an effective method for improving PHA biosynthesis by many microorganisms [43][44][45][46][47] . However, the use of RSM to improve P(3HB-co-3HHx) production has been limited.
The application of batch fermentation by various bacteria to increase PHA biosynthesis in the bioreactor has been reported [48][49][50][51][52] . However, in this present study, P(3HB-co-3HHx) production cannot be enhanced using this approach. Only the 3HHx monomer's composition was increased to 5 mol%, which may be related to the reduced harvesting period. In addition, batch cultivations are easy to operate yet have poor intrinsic productivity due to the restricting concentration of carbon and nitrogen supplies at the beginning of fermentation 53 . In contrast, the fed-batch fermentation method produces a high cell concentration, improves productivity, and reduces substrate or end-product inhibition 54 .
In this study, the P(3HB-co-3HHx) copolymer containing 5 mol% 3HHx monomer composition was synthesized by the engineered strain, C. necator PHB − 4/pBBR_CnPro-phaC Rp using CPKO and sodium hexanoate as a carbon and precursor, respectively. The copolymer was extracted and characterized to understand the structural and thermal characteristics of the copolymer for further use. The 1 H NMR spectrum confirmed the existence of 3HHx monomer in P(3HB-co-3HHx) copolymers synthesized by C. necator PHB − 4/pBBR_CnPro-phaC Rp . It was similar to the 1 H NMR spectra reported by Wong et al. 30 and Bhubalan et al. 55 . Besides, according to the 3HHx monomer composition calculation from 1 H NMR spectra, this polymer contains 5-6 mol% 3HHx monomer, similar to the GC analysis results. The FTIR spectra of copolymer demonstrated the characteristic absorption peak of P(3HB-co-3HHx) copolymers at 1720.98 and 1269.35 cm −1 , which corresponds to the stretching vibration of the carbonyl (C=O) ester bond and asymmetric C-O-C stretching vibration, respectively 31,32 . These results established that the produced copolymer was P(3HB-co-3HHx).
Thermal properties of the P(3HB-co-5 mol% 3HHx) copolymer produced by C. necator PHB − 4/pBBR_CnPro-phaC Rp were analyzed using DSC and TGA. The DSC thermogram of extracted copolymer revealed two melting temperatures (T m1 and. T m2 ) at approximately 129 and 144 °C. Two melting temperatures for mcl-PHAs are detectable, which may be related back to the formation of two different crystal phases (phase I and phase II) 56 . The T c , T g and T d of the copolymer were 89, 1.6 and 260.6 °C, respectively. These results are similar to the previously studied by Murugan et al. 42 . They have reported that the T m and T g of P(3HB-co-4 mol% 3HHx) produced from C. necator Re2058/pCB113 were 164 and − 1 °C, respectively.
The P(3HB) T d was reported at 280 °C [56][57][58] . The T d copolymer in this study was lower due to the incorporation of the 3HHx monomer. In general, P(3HB-co-3HHx) copolymers had a lower T m and T d than P(3HB), although there was no discernible association between these characteristics and the 3HHx molar fractions 22 .

Conclusions
In this study, the production of the P(3HB-co-3HHx) copolymers by an engineered strain of C. necator PHB − 4/ pBBR_CnPro-phaC Rp was improved using RSM. Under RSM optimum conditions, this strain can produce 3.6 ± 0.4 of P(3HB-co-3HHx) containing 4 mol% 3HHx compositions. Compared to the unoptimized condition, the optimized medium can improve the production of P(3HB-co-3HHx) and 3HHx monomer composition by 1.2 and twofold, respectively. Besides, Interestingly, the 3HHx monomer composition was enhanced to 5 mol% when operating the fermentation in a 10 L stirred-tank bioreactor, which was 2.5-fold higher than the unoptimized condition. The functional group and chemical structure results verified the polymer as P(3HB-co-3HHx), and the produced polymer's thermal properties were similar to industrial P(3HB-co-3HHx).

Materials and methods
Bacterial strain and inoculum preparation. The recombinant strain C. necator PHB − 4/pBBR_CnPro-phaC Rp was cultivated as described by Trakunjae et al. 24 . Briefly, the bacterial strain was cultured on nutrient-rich (NR) agar supplemented with 50 µg/mL kanamycin at 30 °C for 24 h. Then, three full loops of a bacterial colony were transferred in NR medium supplemented with 50 µg/mL kanamycin to prepare the bacterial inoculum. After that, incubated the inoculum flasks at 30 °C with shaking of 200 rpm for 8 h or until the optical density (OD600) reached 4. www.nature.com/scientificreports/ Biosynthesis of P(3HB-co-3HHx) copolymers. The 3% v/v of C. necator PHB − 4/pBBR_CnPro-phaC Rp inoculum was transferred into the P(3HB-co-3HHx) production medium. The mineral medium (MM) for P(3HB-co-3HHx) production consisted of 0.45 g/L of K 2 SO 4 , 4.6 g/L of Na 2 HPO 4 , 4.0 g/L of NaH 2 PO 4 , 0.54 g/L of CO(NH 2 ) 2 [Urea], 0.39 g/L of MgSO 4 , 0.062 g/L of CaCl 2 and 1 mL/L of trace element (TE) solution 59  Harvesting of bacterial cells. The bacterial cells were harvested by centrifugation at 8,000 rpm, 4 °C for 10 min. After that, the cell pellets were washed with distilled water (DW), followed by a mixed solution of DW and hexane in a ratio of 1:1 to remove the oil residues. Next, the cell pellets were rewashed with DW to remove the hexane remains and transferred to a pre-weighed bijoux bottle. Then, the bottles containing bacterial cell pellets were frozen at − 20 °C overnight and lyophilized using a freeze-dryer until completely dry. Finally, the weight of the lyophilized cell was recorded in g/L. At the same time, the PHA content and monomer composition were examined by gas chromatography (GC) analysis.
Optimization of P(3HB-co-3HHx) copolymers using RSM. RSM, a practical modeling method, is a set of statistical and mathematical tools for creating experiments and optimizing the influence process variables 60 .
In this study, the production of P(3HB-co-3HHx) copolymers was enhanced using the RSM based on central composite design (CCD). It is commonly used to construct a second-order polynomial for the response variables without a full factorial design of experiments. Three significant factors were used in this study, i.e., CPKO (g/L) (X1), sodium hexanoate (g/L) (X2), and cultivation time (h) (X3). Each variable was coded at five levels (1.68, 1, 0, + 1, and + 1.68) based on the CCD design to define the characteristics of the response surface in the optimal region. A total of twenty fermentation runs were designed according to Eq. (1), including five replicated fermentation runs at the center points.
where k is the number of independent variables and n0 is the number of repetitions of experiments at the center point The coded and actual levels of the significant factors are presented in Table 3. The design matrix of the tested fermentation runs is demonstrated in Table 1 where Y is the predictive measured response; X i and X j are the independent variables; β 0 represents the intercept; and β i , β ii , and β lj are the regression coefficients of the model 61 . The generated model for three independent variables is shown in Eq. (3).

Validation of the RSM model.
The values of the three tested factors, CPKO, sodium hexanoate, and cultivation time, were chosen randomly from the design space to verify the P(3HB-co-3HHx) production by C. necator PHB − 4/pBBR_CnPro-phaC Rp in a shake flask model. In this experiment, the other components of the medium were at fixed levels.
(1) Total number of experiments = k 2 + 2k + n0 www.nature.com/scientificreports/ Scaling up of P(3HB-co-3HHx) production in a 10L bioreactor. Fermentation was carried out in a 10 L stirred-tank bioreactor (Model MDFT-N-10L, Marubishi, Japan) to improve the production of P(3HBco-3HHx) by C. necator PHB − 4/pBBR_CnPro-phaC Rp . The 3% v/v of bacterial inoculum was transferred to the bioreactor containing 6 L of optimized media. Batch cultivation was carried out at 30 °C with initial pH and agitation speeds of 6.8 and 200 rpm, respectively. The pH of the culture broth was maintained at pH 6.8 during the fermentation by adding HPO 3 or NaOH using a pH controller. The airflow rate was fixed at 0.25 vvm. The cell biomass and P(3HB-co-3HHx) production were evaluated every 6 h during 48 h of fermentation. The fermentations were performed in triplicates and average values were determined. P(3HB-co-3HHx) copolymers extraction and purification. The 10 g of freeze-dried cells were dissolved in 1 L chloroform and stirred for 3-5 days at room temperature to extract the P(3HB-co-3HHx) copolymers. Then, the cell debris was removed by filtering the bacterial cell suspension using filter paper (Whatman No. 1). After that, the P(3HB-co-3HHx) dissolved chloroform solution was evaporated to approximately 100 mL using a rotary evaporator. The evaporated solution was subsequently added drop by drop to 100 mL of ice-cold methanol and stirred for 1 h. Finally, the purified polymer was separated by filtration using 0.45 µm PTFE membrane and air dried for 3-5 days 44 before being used for further experiments.
Characterization of P(3HB-co-3HHx) copolymers. Proton nuclear magnetic resonance ( 1 H NMR) spectroscopy is a simple technique to investigate PHA polymer composition. In this study, the purified P(3HBco-3HHx) copolymers were dissolved in deuterated chloroform (CDCl 3 ) at 25 mg/mL to apply for NMR analysis. The solution-state 1 H NMR was carried out on a Jeol JNM-ECZ-400R/S1 spectrophotometer (JEOL, Ltd., Tokyo, Japan) resonating at 500 MHz. The chemical shifts were referred to the tetramethylsilane (TMS). At the same time, adamantane was used as an external standard. The functional groups of purified P(3HB-co-3HHx) copolymers were detected by Fourier transform IR (FTIR) spectroscopy. The FTIR analysis was performed using an FTIR spectrometer (Thermo Scientific Nicolet IR200, Waltham, MA, USA). The 128 scans were composed in attenuated total reflection (ATR) mode. Besides, the spectra were achieved in the range of 4000 to 400 cm −1 with a resolution of 4 cm −1 .
The purified P(3HB-co-3HHx) copolymers were analyzed for their thermal properties using differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). DSC analysis was analyzed by DSC25 (TA instruments, New Castle, DE, USA) using a nitrogen flow rate of 30 mL/min. Around 3-5 mg of purified P(3HB-co-3HHx) copolymers were filled into a Tzero Aluminum Hermetic pan, covered, and heated from 25 to 200 °C at a heating rate of 15 °C/min. The melted samples were then maintained at 200 °C for 2 min and rapid reduction to − 40 °C. Finally, they were repeatedly heated from − 40 to 200 °C at a heating rate of 15 °C/min. The melting temperature (T m ), crystallization temperature (T c ), and glass transition temperature (T g ) were detected and analyzed from the DSC thermogram. For TGA analysis, approximately 5 mg of the purified P(3HB-co-3HHx) copolymers were filled in an aluminum pan and analyzed using Pyris 1 TGA instrument (Perkin Elmer, USA). The heating temperature was set from 30 to 900 °C at a heating rate of 20 °C/min under a nitrogen atmosphere.
Analysis of dry cell weight (DCW). The determination of DCW was modified from Trakunjae et al. 24 .
Briefly, 1 mL of cell culture suspension was transferred into the pre-weighed Eppendorf tubes and centrifuged at 8,000 rpm for 10 min. Then, the harvested cells were washed with distilled water, followed by a mixed solution of DW and hexane in a ratio of 1:1 to remove the oil residues. Then washed with DW to remove hexane remains and centrifuged at 8,000 rpm for 10 min. Next, the obtained cell pellets were frozen at − 20 °C overnight and lyophilized using a freeze-dryer for 2-3 days. Finally, the Eppendorf tubes containing lyophilized cells were weighed to verify stability and calculated the DCW in g/L.
Analysis of PHA content. The PHA content and monomers composition were analyzed using the methanolysis technique following Braunegg et al. 62 . Briefly, 15-20 mg of lyophilized cells were added to the test tube, followed by 2 mL of chloroform and methanolysis solution (mixture of 85% v/v of methanol and 15% w/v H 2 SO 4 ). The tubes were heated at 100 ̊ C for 180 min, then cooled at room temperature. After that, 1 mL of DW was added into the tubes and mixed vigorously for 1 min using a vortex mixer. The chloroform-rich PHA in the bottom layer was collected using a pasture pipette. Then, remove the water residues using Na 2 SO 4 . The mixture solution of 500 mL of chloroform-rich PHA solution and 500 mL of 0.2% (v/v) caprylic methyl ester (CME) (Internal standard) was prepared for GC analysis. The analysis was performed using Shimadzu GC-2014 plus (Shimadzu, Japan) supplied with Restek RTX-1 column (Restek, USA) and flame ionization detector (FID). The 2.0 µL of the prepared sample solution was injected into the GC machine. Nitrogen was used as a carrier gas for GC analysis. Besides, the injector and detector temperatures were set at 270 °C and 280 °C, respectively. Statistical analysis. All experimental data were described as mean ± standard error. The statistical analysis was carried out by SPSS statistics 17.0 software (SPSS for Windows, SPSS Inc., Chicago, IL, USA). Experimental responses were examined using a two-way analysis of variance (ANOVA). Each model term's linear, quadratic, and interaction regression coefficients were calculated using the F-value at a probability (P) < 0.05. In addition, the statistical significance of each term in the polynomial was analyzed, and all coefficients were investigated using Design-Expert® v7.0.0 software (Stat-Ease, Inc. MN, USA). www.nature.com/scientificreports/